Bioreactor and Method

ABSTRACT

The bioreactor is for use in performing biological and/or biochemical reactions and includes a vessel, an agitator, a reaction assembly, and a harvesting outlet. The vessel of the bioreactor includes several ports including a mixing port, a reaction port, and a harvesting port. The agitator extends through the mixing port into the vessel while the harvesting outlet extends through the harvesting port and permits the withdrawal of reaction medium to another vessel. The reaction assembly extends through the reaction port into the vessel and has multiple components including a gas conduit adapted to introduce gas into a reaction medium in the vessel, a sampling device adapted to remove a portion of the reaction medium from the vessel without contamination of the remaining reaction medium, and an introduction conduit permitting the introduction of at least the reaction medium into the vessel.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to bioreactors for performing biologicaland/or biochemical reactions. More specifically, the present inventionrelates to a bioreactor for culturing cells (e.g., biological samples)and for use in the production of medical materials such as hormones,enzymes, antibodies, vaccines, and drugs. The invention further relatesto an improved bioreactor that is easy to assemble and maintainssterility throughout the culturing/fermentation operation, and providesfor sterile transfer of material to larger vessels.

2. Description of Related Art

In the medical field, it is often necessary to cultivate biologicalsamples for further study of cell structures. Such culturing is oftencarried out in a bioreactor, which is essentially a sterile vessel thatprovides internal sealed conditions suitable for cell growth.Bioreactors are also used in the production of medical materials such ashormones, enzymes, antibodies, vaccines, and drugs. Typically, thebioreactor produces batch quantities of the desired material to “seed”larger vessels for further processing. These larger vessels areessentially large bioreactors, which continue the culturing/productionprocess. The “smaller” bioreactors are used to produce an effectiveamount of material of sufficient concentration such that the materialcan continue culturing/fermentation in the larger vessel. The largerbioreactor vessels have the increased capacity necessary to produceincreased quantities of the desired material for study purposes or forfurther processing into a final product such as a vaccine or drug. Thesmaller bioreactors, which feed larger vessels, are limited inproduction capability because, for a given amount of reaction mediumintroduced into the smaller bioreactor, the bioreactor can generate onlya finite amount of product. As a result, transfer of a “seed” product toa larger vessel is necessary for further production.

Generally, three basic types of bioreactors are used in the medicalfield for producing biological and/or biochemical reactions, namely, astationary type, a shaking type, and a rotating type. In stationarybioreactors, a fixedly settled reaction vessel, such as an Erlenmeyerflask, is employed in which a stirrer is provided and which operates toproduce a generally horizontal rotary movement to cause agitation of thereaction mixture. This stirring action increases the heat transfer andoxygen transfer rate. While this type of bioreactor permits economicalrunning of reactions, it typically cannot operate with a highly viscoussubstrate due to insufficient driving torque of the stirrer, and theheat and oxygen transfer are mass transfer limited. In this type ofbioreactor, substrates with densities different from that of thesolvent, (i.e., the reaction medium), tend to float up or settle downwhich causes separation of the reaction mixture.

In shaking type bioreactors, a shaking culture using test tubes as theculture vessel has been widely used in the medical field. In this typeof system, the reaction is accelerated by reciprocating horizontalmovement of the culture vessel. This type of bioreactor also does notoperate well with highly viscous mediums and also suffers the same masstransfer and separation problems as the stationary type bioreactordiscussed previously.

Rotating type bioreactors are most commonly used in the medical field.In rotating bioreactors, the reaction medium is subjected to agitationby a rotating apparatus inserted into the vessel. An example of arotating bioreactor is disclosed in U.S. Pat. No. 5,587,298 to Horiganeet al. This patent discloses a bioreactor comprised of a reaction vesseldefining a reaction chamber which is surrounded by a water jacket formaintaining a predetermined constant temperature. The reaction chamberis provided in the vicinity of the inner wall surface of the reactionvessel. A pair of upright screws is disposed in a side-by-side andadjoining relationship and is used to agitate the reaction mixture. Inparticular, each screw has a screw thread or a helical fin which engagesthe corresponding screw thread on the adjacent screw to introduceagitation into the reaction mixture. The reaction mixture is subjectedto uniform agitation and to a milling and sieving action in the areawhere the screw threads are engaged.

Another example of a rotating bioreactor is disclosed in U.S. Pat. No.4,636,675 to Freedman et al. This patent discloses, in particular, animproved agitator for a bioreactor vessel. The agitator includes ahollow body assembly having an open end and a closed end and a tubularstructure communicating with the hollow body assembly at a locationspaced from the open end. The tubular structure has an exit openingoriented such that rotational movement of the agitator and the tubularstructure causes a suction or draft at the exit opening to cause flow offluid from the open end of the hollow body assembly through the hollowbody assembly and out through the tubular structure exit opening. Theagitator is driven by a magnetic drive motor.

Another important aspect of bioreactor systems is the ability tointroduce oxygen or other gases to the cell cultures to assist in cellgrowth to increase the oxygen transfer rate. Aeration of a cell cultureor reaction medium is typically achieved by conventional means such asbubbling gases through the fluid by placing a delivery tube in the fluidwith an exit near the bottom thereof. However, bubbling air or othergases into cell cultures in this manner can damage the cultures and itis advantageous to minimize bubble size to reduce possible damage to theculture. Typically, a sparger is used to reduce bubble size and islocated at the end of the delivery tube. One example of a sparger foruse in a bioreactor is disclosed by U.S. Pat. No. 4,727,040 to Freedmanet al. This patent discloses a sparger, which includes a narrow,vertically extending outer chamber defined by a solid wall and a wallformed from a screen. Air or other gas is then forced under pressurethrough a tube to the bottom of the outer chamber and from there to adistribution member having apertures distributed along the lower regionof the outer chamber for the release of the gas upwardly into thechamber. The top of the outer chamber is provided with exit openingswhich communicate to a point above the surface of the medium. The gasbubbles pass through the column of fluid in the outer chamber, a portionwhich passes through the screen in the form of fine bubbles to reducebubble size.

With the foregoing background in mind, there is room for improvement inbioreactor design and construction particularly in the area ofbioreactor assembly, operation, and the ability to transfer contents tolarger reaction vessels in a sterile manner for further processing.

SUMMARY OF THE INVENTION

The bioreactor is intended for use in performing biological and/orbiochemical reactions and includes a vessel, an agitator, a reactionassembly, and a harvesting outlet. The vessel of the bioreactor includesseveral ports including a mixing port, a reaction port, and a harvestingport. The agitator extends through the mixing port into the vessel whilethe harvesting outlet extends through the harvesting port and permitsthe withdrawal of reaction medium to another vessel, which may be partof a bioreactor system. The reaction assembly extends through thereaction port into the vessel and has multiple components including agas conduit adapted to introduce gas into a reaction medium in thevessel, a sampling device adapted to remove a portion of the reactionmedium from the vessel without contamination of the remaining reactionmedium, and an introduction conduit permitting the introduction of atleast the reaction medium into the vessel.

The bioreactor may further include a water jacket adapted to regulatethe temperature of the reaction medium. The gas conduit may be adaptedto aerate the reaction medium. A sparger may be provided in the gasconduit of the reaction assembly to limit the bubble size of the gasintroduced into the vessel. The agitator may extend through a coversealing the mixing port. The agitator may be adapted to be coupled to anexternal drive device, for example, a motor.

The sampling device may comprise a collection conduit extending into thevessel, a sampling valve positioned in the collection conduit anddisposed outside the vessel, and an extractor associated with thecollection conduit and disposed outside of the vessel. The extractor maybe adapted to remove a sample portion of reaction medium through thecollection conduit. A discharge outlet may be provided for releasing thesample portion of reaction medium from the collection conduit outside ofthe vessel without contamination of the remaining reaction medium. Theextractor may further comprise a vacuum generating portion adapted todecrease pressurization in the collection conduit, and a filter elementprovided fluidly between the vacuum generating portion and thecollection conduit.

The bioreactor may be used as part of a bioreactor system for performingbiological and/or biochemical reactions. In such a system, the systemincludes a seed bioreactor comprising a vessel, an agitator, a reactionassembly, and a harvesting outlet. The vessel of the bioreactor includesseveral ports including a mixing port, a reaction port, and a harvestingport. The agitator extends through the mixing port into the vessel whilethe harvesting outlet extends through the harvesting port and permitsthe withdrawal of reaction medium. The reaction assembly extends throughthe reaction port into the vessel and has multiple components includinga gas conduit adapted to introduce gas into a reaction medium in thevessel, a sampling device adapted to remove a portion of the reactionmedium from the vessel without contamination of the remaining reactionmedium, and an introduction conduit permitting the introduction of atleast the reaction medium into the vessel. The seed bioreactor may beused to “seed” a main (e.g., larger) bioreactor with reaction medium forcontinuing the biological and/or biochemical reaction process. Such amain bioreactor is adapted to be placed in fluid communication with thevessel via the harvesting outlet and typically includes an inlet port.The main bioreactor may be placed in fluid communication with the vesselvia a sanitary connection between the harvesting outlet and the inletport.

Another aspect of the invention is related to a method of assembling abioreactor adapted to perform biological and/or biochemical reactions.The method includes providing a vessel comprising a mixing port, areaction port, and a harvesting port; inserting an agitator into thevessel through the mixing port, with the agitator adapted to stir areaction medium in the vessel; sealing the mixing port with a cover,with the agitator extending through the cover; sealing the reaction portwith a reaction assembly extending through the reaction port and intothe vessel; and sealing the harvesting port with a harvesting outletadapted to permit the sterile withdrawal and transfer of reaction mediumto another vessel. The reaction assembly generally comprises a gasconduit adapted to introduce gas into the vessel, a sampling deviceadapted to remove a portion of the reaction medium from the vesselwithout contamination of the remaining reaction medium, and anintroduction conduit permitting the introduction of at least thereaction medium into the vessel.

A further aspect of the invention relates to a method of preparingbiological and/or biochemical reactants, generally comprising the stepsof providing a seed bioreactor comprising a vessel having a mixing port,a reaction port, and a harvesting port. The method further includesintroducing reactants (i.e., reaction medium) into the vessel through anintroduction conduit extending through the reaction port; introducinggas into the reaction medium through a gas conduit extending through thereaction port; agitating the reaction medium with a mixer extendingthrough the mixing port; and transferring at least a portion of theprocessed reaction medium to a main bioreactor through the harvestingport.

Further details and advantages of the invention will become clear uponreading the following detailed description in conjunction with theaccompanying drawing figures, wherein like parts are identified withlike reference numerals throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded and partially perspective view of a bioreactorpursuant to an embodiment of the present invention.

FIG. 2 is an assembled and partially perspective view of the bioreactorof FIG. 1.

FIG. 3 is a schematic diagram showing the bioreactor of FIG. 2 as partof a system wherein the bioreactor is a seed bioreactor for largerbioreactor devices.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of the description hereinafter, spatial orientation terms,if used, shall relate to an embodiment of the invention as it isoriented in the accompanying drawing figures or otherwise described inthe following description of the invention. However, it is to beunderstood that the invention embodiments described hereinafter mayassume many alternative variations and configurations. It is also to beunderstood that the specific devices illustrated in the accompanyingdrawing figures and described herein are simply exemplary to theinvention and should not be considered as limiting.

Referring generally to FIGS. 1-2, an embodiment of a “seed” bioreactor10 for performing biological and/or biochemical reactions is shown.Bioreactor 10 is generally defined by a reaction vessel 20 which isillustrated as having a cylindrical shape for exemplary purposes.Reaction vessel 20 is intended to encompass and retain a reaction mediumR which is to undergo a biological and/or biochemical reaction.Accordingly, it is desirable that vessel 20 be made of a material thatis inert to reaction medium R, for example, stainless steel or asuitably lined metallic vessel, or possibly an inert plastic material.This type of construction is desirable to prevent introducing unwantedsubstances into reaction medium R. Reaction vessel 20 is of adouble-walled construction comprising an outer wall 22 a and an innerwall 22 b. Outer wall 22 a and inner wall 22 b define spacingtherebetween for introduction of water thereby forming a water jacket 24within the wall of reaction vessel 20. Water jacket 24 acts as atemperature controlling device for maintaining/regulating thetemperature of reaction medium R and, thus, reaction vessel 20generally, for example, at a predetermined temperature. Water istypically used as the temperature controlling medium and is passedthrough water jacket 24 surrounding reaction vessel 20 and may becirculated at a constant temperature from a constant temperature bath,as an example.

Water jacket 24 has an inlet 26 and an outlet 28 for passing watertherethrough to maintain the temperature of reaction medium R andreaction vessel 20. In the illustrated embodiment, inlet 26 of waterjacket 24 may be positioned at a lower portion of outer wall 22 a andoutlet 28 may be positioned at an upper portion of outer wall 22 a onthe opposite side thereof. Accordingly, in order to provide a constanttemperature to reaction vessel 20, both inlet 26 and outlet 28 areconnected to a constant temperature bath via a circulation line (notshown) and a circulation pump (not shown). Water jacket 24 may becontrolled so as to maintain constant temperature by temperature signalsfrom some form of heat sensor connected to an external control unit (notshown) such as a computer. Conventional circulation connections betweeninlet 26 and outlet 28 of water jacket 24 may be used to create a closedloop system. If desired, inlet 26 may be positioned at an upper portionof outer wall 22 a while outlet 28 is positioned at a lower portion toencourage the venting of air from water jacket 24 and to minimizeincomplete filling of water jacket 24.

Reaction vessel 20 defines several ports or openings, including a mixingport 30, a reaction port 60, and a harvesting port 90. These ports 30,60, 90 are covered by respective covering or closure devices which arediscussed herein. These closure devices seal ports 30, 60, 90 to form anenclosed environment E or space within reaction vessel 20 suitable forbiological and/or biochemical reactions. Mixing port 30 supports amixing apparatus 32 which effectuates a generally fluid-tight seal withmixing port 30 to close mixing port 30 and, further, treatment ofreaction medium R as discussed herein. Mixing apparatus 32 includes anagitator 34 which extends through mixing port 30 into reaction vessel20. Agitator 34 comprises a shaft 36 extending into reaction vessel 20and a paddle-shaped impeller 38 disposed in the enclosed environment Edefined by reaction vessel 20 for stirring or agitating reaction mediumR received in reaction vessel 20. Impeller 38 may take otherconventional forms known within the bioreactor/fermenter art. Agitator34 desirably extends into reaction vessel 20 to ensure sufficientagitation/stirring of reaction medium R when in operation and impeller38 is sufficiently spaced from inner wall 22 b of reaction vessel 20 toensure that sufficient agitation/stirring/oxygen transfer rate ofreaction medium R can occur. Agitator 34 is supported in a generallyvertical or upright position by a mounting and sealing apparatus 40engaged in mixing port 30. However, agitator 34 remains rotatablethrough mounting and sealing apparatus 40. Mounting and sealingapparatus 40 supports agitator 34 while maintaining a generallyfluid-tight seal of mixing port 30 to prevent any contamination ofreaction medium R in reaction vessel 20.

For driving agitator 34, a motor M or other driving device may beemployed, typically outside of reaction vessel 20. Motor M may have anysuitable transmission mechanism for transmitting driving power toagitator 34. Reaction medium R is mixed when the torque of motor M istransmitted to agitator shaft 36 and impeller 38 through the fluid-tightseal formed by mounting and sealing apparatus 40. Further, drivingdevice (or motor M) may be actuated by a command signal from a controlunit (not shown) to cause agitator 34 to rotate. By controlling theagitation condition in reaction vessel 20, it is possible to employvarious states or speeds of agitation to assist in the reaction processof reaction medium R in reaction vessel 20.

Mounting and sealing apparatus 40 (hereinafter “mounting apparatus 40”)comprises a sealing “first” cover 42 adapted to enclose andsubstantially seal mixing port 30. Cover 42 is adapted to engage or seaton/in mixing port 30 and, in particular, seal against a first rim 44 ofreaction vessel 20 which defines mixing port 30. A gasket may also beused to ensure a generally fluid-tight characteristic between cover 42and rim 44. Cover 42 is secured to reaction vessel 20 and on rim 44 inparticular by a securing ring 46, thereby closing and substantiallysealing mixing port 30. In one embodiment, ring 46 is internallythreaded to engage an externally threaded portion 48 of reaction vessel20 below rim 44. The threaded engagement between these elements securesthe position of cover 42 on rim 44 and, further, the generallyfluid-tight characteristic between cover 42 and rim 44. Agitator 34extends through a central opening 49 in cover 42 and a generallyfluid-tight seal is provided between agitator 34 and central opening 49.If desired, a nut head (not shown) or similar structure may beintegrated or formed with the body of rim 44 to allow easy threadedengagement of rim 44 to externally threaded portion 48.

Cover 42 defines one or more vent openings 50 which accept ventstructures or valves 52. Vent valves 52 may be threadedly engaged invent openings 50 which allow venting of the enclosed environment Edefined by reaction vessel 20. Vent valves 52 may be one-way valves thatprevent external contamination from entering enclosed environment E.Additionally, filter elements 54 may be placed in fluid communicationwith and downstream of vent valves 52 and may be connected to ventvalves 52 by tubing 56 to substantially prevent any contamination fromreaction medium R from reaching the external atmosphere. Vent valves 52may be used, for example, to ensure that reaction medium R in reactionvessel 20 is not compromised by an excess build-up of gasses introducedinto reaction vessel 20. Finally, agitator 34 may be connected to an endor coupling plate 58 which is adapted for connection to the drive outputof motor M for imparting rotational driving force to agitator 34 andagitator shaft 36 and impeller 38 in particular.

As indicated previously, reaction vessel 20 defines or includes reactionport 60. A reaction assembly 62 is inserted in reaction port 60 andeffectuates a generally fluid-tight seal with reaction port 60 to closeand seal reaction port 60. Reaction assembly 62 is provided as theinterface device for introducing reaction medium R and reaction enablingmaterials and substances into reaction vessel 20, as well as forwithdrawing or removing quantities of reaction medium R from reactionvessel 20 for sampling and testing purposes. Generally, reactionassembly 62 includes a gas introduction conduit 64, a reactantintroduction conduit 66, and a sampling device 68. Reaction assembly 62generally extends through reaction port 60 and into reaction vessel 20.

A sealing “second” cover 72 is provided as part of reaction assembly 62and is used to enclose and substantially seal reaction port 60. Cover 72is adapted to engage or seat on/in reaction port 60 and, in particular,seal against a second rim 74 of reaction vessel 20 which definesreaction port 60. Cover 72 is secured to reaction vessel 20 and on rim74 in particular by a “second” securing ring 76, thereby closing andsubstantially sealing reaction port 60. A gasket or O-ring G is providedbetween cover 72 and ring 76 to ensure a generally fluid-tightcharacteristic between cover 72 and rim 74. In one embodiment, ring 76is internally threaded to engage an externally threaded portion 78 ofreaction vessel 20 below (i.e., adjacent) rim 74. The threadedengagement between these elements secures the position of cover 72 onrim 74 and, further, the generally fluid-tight characteristic betweencover 72 and rim 74. Although the mixing and reaction ports 30, 60 areshown with respective covers 42, 72 threadably secured to reactionvessel rims 44, 74 to establish a generally fluid-tight construction, itis also possible to permanently affix the respective covers 42, 72 toreaction vessel rims 44, 74. Cover 72 defines several openings 80 forpassage of gas introduction conduit 64, introduction conduit 66, and acollection conduit 82 associated with sampling device 68. Conduits 64,66, 82 are each disposed in the respective openings 80 in a generallyfluid-tight manner. If desired, a nut head (not shown) or similarstructure may be integrated or formed with the body of rim 74 to alloweasy threaded engagement of rim 74 to externally threaded portion 78.

Further, a filter element 84 may be associated with gas introductionconduit 64 via tubing 86 so that sterile-filtered gas may be introducedin reaction medium R by the gas introduction conduit 64 and to preventany contamination from reaching reaction medium R from the external gassupply source (not shown). Gas introduction conduit 64 is adapted tointroduce gas, for example, oxygen or air, into reaction medium R inreaction vessel 20 to assist in the biochemical process occurring in thereaction medium R. For example, if reaction medium R contains cells forculturing, oxygen is necessary for cell growth and oxygen or air may beintroduced via gas introduction conduit 64 to support the cell growth.Further, the introduction of carbon dioxide may be necessary to formessential ions for growing cells and is another example of a gas thatmay be introduced into reaction medium R via gas introduction conduit64. Because reaction vessel 20 is designed to maintain a generallyfluid-tight and sealed condition, an anaerobic gas may alternatively oradditionally be introduced into reaction medium R and may be used toaerate reaction medium R. Gas introduction conduit 64 may be connectedto a gas bomb (not shown), for example, as a gas supply source for thegas introduction conduit 64 and a metering or control valve may beassociated with gas introduction 64 to control the delivery of gas intoreaction medium R. Finally, a sparger 88 is disposed at the end of gasintroduction conduit 64 for distributing gas into reaction medium R and,desirably, to reduce size of the gas bubbles introduced into thereaction medium R to prevent damage to cells or other bio-matter in thereaction medium R. Although sparger 88 is illustrated in the figures fordistributing the introduced gas into reaction medium R, other equivalentmeans for introducing, distributing, and/or aerating gas into reactionmedium R and enclosed environment E generally may be substituted inplace of sparger 88. Examples of such equivalent devices include devicescapable of caged sparging, medium perfusion, membrane diffusion, etc.

As indicated previously, reaction vessel 20 defines or includesharvesting port 90. A harvesting outlet assembly 92 (hereinafter“harvesting outlet 92”) is inserted into and disposed in harvesting port90 and is used to “harvest” or withdraw reaction medium R from reactionvessel 20, typically in large volumes, and transfer the same to one ormore larger bioreactor vessels for further culturing/fermentation (i.e.,scale-up processing). Harvesting outlet 92 includes a harvesting conduit94 which is inserted through harvesting port 90 and extends intoreaction vessel 20 to be in contact with reaction medium R in enclosedenvironment E defined by reaction vessel 20 for removing all or aportion of the reaction medium R to a downstream process. A first orinternal end 96 of harvesting conduit 94 is disposed in reaction vessel20 and is in contact with reaction medium R in reaction vessel 20. Asecond or external end 98 of harvesting conduit 94 desirably terminatesin a generally planar sanitary coupling 100 of conventional design. Thefunction and use of sanitary coupling 100 is discussed herein.

Harvesting outlet 92 is desirably adapted to effectuate a generallyfluid-tight seal with harvesting port 90 to close and seal harvestingport 90. Harvesting outlet 92 comprises a sealing “third” cover 102disposed about harvesting conduit 94, or affixed thereto, and whichextends radially outward from harvesting conduit 94. Cover 102 is usedto engage and/or seat on/in harvesting port 90 and, in particular, sealagainst a third rim 104 of reaction vessel 20 defining the harvestingport 90. Cover 102 is then secured to reaction vessel 20 and on rim 104in particular by a “third” securing ring 112, thereby enclosing andsubstantially sealing the harvesting port 90. Accordingly, in oneembodiment, ring 112 may be an annular structure that is internallythreaded and rotatably received about harvesting conduit 94 onexternally threaded portion 108 below (i.e., adjacent) rim 104. A gasketor O-ring 106 is provided between ring 112 and rim 104 to ensure agenerally fluid-tight characteristic between cover 102 and rim 104.Thus, ring 112 is similar in structure and operation to rings 46, 76discussed previously. While a threaded engagement between rim 112 andexternally threaded portion 108 is shown in the figures, it is alsopossible to permanently affix ring 112 to reaction vessel rim 104. Ifdesired, a nut head (not shown) or similar structure may be integratedor formed with the body of rim 112 to allow easy threaded engagement ofrim 112 to externally threaded portion 108.

As indicated previously, harvesting conduit 94 may terminate at externalend 98 with sanitary coupling 100 which may be used to connectharvesting outlet 92 with another vessel or an apparatus used to removeall or a portion of reaction medium R from reaction vessel 20.Harvesting conduit 94 (and sanitary coupling 100) may alternatively besealed and/or covered (not shown) to prevent contamination of theenclosed environment E of the reaction vessel 20. Sanitary coupling 100may also be connected to a sanitary valve 110 via a sanitary clamp (notshown) or other suitable union. Sanitary valve 110 allows metered orcontrolled withdrawal of reaction medium R from reaction vessel 20 andcomprises a valve body 114 and an actuator 116 or control associatedwith valve body 114. Actuator 116 controls operation of valve body 114,for example, by opening and closing valve body 114 to permit thecontrolled withdrawal of reaction medium R from reaction vessel 20.Valve body 114 desirably itself includes sanitary inlet and outletcouplings 118, 120 for engaging the sanitary coupling 100 at externalend 98 of harvesting conduit 94 and a downstream apparatus to which thereaction medium R is to be supplied such as a larger bioreactor vesselfor continued processing of the reaction medium R as discussed herein inconnection with FIG. 3.

For supplying reaction vessel 20 with reactants which form and/orpromote culturing/fermentation of reaction medium R, reaction assembly62 includes introduction conduit 66. Introduction conduit 66 ispositioned to extend outward from ring 76 and is typically connected toa reactant source for supplying reactants which form and/or promoteculturing/fermentation of reaction medium R. However, in the eventintroduction conduit 66 is not connected to a reactant source in somemanner, the introduction conduit 66 may be sealed and/or covered (notshown) to prevent contamination of the enclosed environment E of thereaction vessel 20. Introduction conduit 66 may include a similarsanitary coupling (not shown) to sanitary coupling 100 which may coverintroduction conduit 66 in the manner discussed previously so thatreactants may be provided to reaction vessel 20 without contamination.Further, reactants could be introduced automatically from, for example,a hopper (not shown) connected to introduction conduit 66. If desired,reactants may be introduced to reaction vessel 20 at a constant rate bydosing a predetermined amount of the desired reactants at desired timeintervals via a feed pump (not shown) connected to introduction conduit66.

As discussed previously, one component of reaction assembly 62 issampling device 68. Sampling device 68 is provided for removing aportion, typically a small amount of reaction medium R from reactionvessel 20, for testing purposes to monitor the culturing/fermentationprocess for completeness or determine whether additions to reactionmedium R are needed to complete the process. Sampling device 68generally includes collection conduit 82, discussed previously, whichextends through reaction port 60 and into reaction vessel 20 to aposition in fluid contact with reaction medium R. A sampling valve 122is positioned outside of reaction vessel 20 and is provided incollection conduit 82 for controlling the outtake of reaction medium Rfrom reaction vessel 20 and to maintain a sterile barrier of reactionmedium R with the external environment. A valve actuator 124 is providedto control operation of sampling valve 122. As an example, samplingvalve 122 may be a cavity filled ball valve or diaphragm valve, althoughany number of other types of aseptic valves may be implemented invarious embodiments depending upon the application for which samplingdevice 68 is to be employed.

A sample extractor 126 is in fluid communication with sampling valve 122provided in collection conduit 82. Sample extractor 126 is locateddownstream of sampling valve 122 and exterior to the reaction vessel 20.Sampling valve 122 and sample extractor 126 are operable to permit auser of bioreactor 10 to selectively remove a small amount of reactionmedium R from reaction vessel 20 for testing and evaluation purposes orother purposes as desired. Sample extractor 126 includes a vacuumgenerating portion 128, such as a simple vacuum bulb, which is in fluidcommunication with collection conduit 82 for producing vacuum (i.e.,negative) pressure within collection conduit 82 to cause removal of asmall sample of reaction medium R from reaction vessel 20 when samplingvalve 122 is in an open position. Sampling device 68 allows removal of asmall amount of reaction medium R when sampling valve 122 in an openposition without contaminating the remaining reaction medium R withinreaction vessel 20, as described herein. Collection conduit 82terminates in a discharge outlet 130 from which the withdrawn sample isextracted to another vessel or container. Discharge outlet 130 may be asimple beveled orifice as shown or could include an on-off valve forcontrolling flow of the sampled reaction medium R or terminating suchflow. A filter element 132 is desirably disposed between vacuumgenerating portion 128 and a port 133 on collection conduit 82 whichconnects the collection conduit 82 to vacuum generating portion 128 toprevent or reduce the possibility of introducing contamination toreaction medium R when sampling valve 122 is in an open state.Conventional tubing 134 is used to complete the fluid connection betweenfilter element 132 and port 133. A sampling housing 136 may be providedaround the discharge end or portion of collection conduit 82 upstream ofdischarge outlet 130 and is provided, for example, to guide the user inconnecting a collection container or vessel to discharge outlet 130.

In operation, a collection container or vessel (not shown) is placed influid connection with discharge outlet 130. A vacuum is created byforcing air out of the sampling housing 136, which is in fluidcommunication with collection conduit 82 and the collection container. Aseal is formed, typically in a generally fluid-tight or sealed manner.Sampling valve 122 is opened and sample extractor 126 is opened and thevacuum generated in the collection conduit 82, the collection containerand vacuum generating portion 128 is released by drawing reaction mediumR through sampling valve 122 and collection conduit 82 and ultimatelyinto the collection container. Thus, sample extractor 126 creates vacuumpressure in collection conduit 82 which withdraws a sample of reactionmedium R from reaction vessel 20 and delivers this sample to dischargeoutlet 130 and, further, the collection container or vessel connectedthereto. Sampling valve 122 may then be operated to a closed positionre-closing collection conduit 82 and again sealing the enclosedenvironment E in reaction vessel 20. A sample of reaction medium R isnow deposited within the collection container and this container may beremoved for testing and evaluation of the collected sample of reactionmedium R.

In order to further enhance the ability to obtain a sterile sample, theportion of the sampling device 68 downstream of sampling valve 122(i.e., sample extractor 126) may be removable/replaceable. Thus, sampleextractor 126 can be removed from sampling valve 122 and cleaned withalcohol or other sterilizing medium to ensure cleanliness outside valve122. Alternatively, another sterile portion of sampling device 68 may beaffixed to sampling valve 122 to reduce the possibility forcontaminating subsequent samples of reaction medium R taken fromreaction vessel 20. The detachability of this downstream portion may beaccomplished in a number of ways including threaded engagement, snapfit, and like simple mechanical connection techniques.

Referring further to FIG. 3 a bioreactor system 200 comprisingbioreactor 10 as a “seed” bioreactor for larger processing vessels isshown. As is known in the medical art, bioreactors are often used forinitial culturing/fermentation of biological and biochemical substances,which are then transferred to larger processing vessels for scale-upprocessing once an effective amount of processed material is present inthe “seed” bioreactor for transfer to the larger vessel. FIG. 3illustrates such a system 200 comprising bioreactor 10 which isconnected by harvesting sanitary valve 110 to an input/introduction lineor conduit 202 of the system 200. Input line 202 typically includes asanitary coupling 203 similar to sanitary coupling 120 on sanitary valve110 and which is connected to sanitary coupling 120 by a sanitary clamp(not shown) or other suitable union. This sanitary coupling 203 on inputline 202 forms the “input port” to input line 202. The union foreffecting a sanitary connection between couplings 120, 230 is typicallysimilar to the union between sanitary coupling 100 on harvesting conduit94 and inlet sanitary coupling 118 on sanitary valve 110. Input line 202is connected to several large or scale-up bioreactor devices, or mainbioreactors 204, 206, 208, via respective main bioreactor input lines210, 212, 214 and control valves V₁, V₂, V₃. As indicated, mainbioreactors 204, 206, 208 provide for performing biological and/orbiochemical reactions on a larger scale than “seed” bioreactor 10 as iscommon in scale-up processes.

Bioreactor 10 is generally assembled as discussed hereinafter. Howeverthe following assembly steps are intended merely as an example of onepossible assembly method for bioreactor 10. Bioreactor 10 is intended toprovide a sterile and sealed enclosed environment for reaction medium Rin reaction vessel 20. Accordingly, the mixing port 30, reaction port60, and harvesting port 90 are each be sealed and sterilized via heat,chemical or other method before the reaction medium R can be asepticallyintroduced into reaction vessel 20 through a sterilizing grade liquidfilter (not shown). Alternatively, if reaction medium R can withstandsterilization via heat, chemical or radiation, reaction medium R can beintroduced into the vessel 20 and sealed prior to total vesselsterilization. Beginning with mixing port 30, mixing apparatus 32 isinserted into mixing port 30 with mounting and sealing apparatus 40enclosing and sealing the mixing port 30. Agitator 34 may bepreassembled with cover 42, and cover 42 may have vent valves 52 securedin openings 50. Likewise, ring 46 may be disposed about agitator 34 sothat the agitator 34, cover 42, and ring 46 are in a preassembled state.This preassembled structure is inserted into mixing port 30 and ring 46is secured to threaded portion 48 below rim 44, which defines mixingport 30, to complete the assembly of mixing apparatus 32 in mixing port30.

Next, reaction assembly 62 may be inserted into reaction port 60 toenclose and seal the reaction port 60. Ideally, reaction assembly 68 ispreassembled with cover 72 supporting conduits 64, 66, 82 and ring 76disposed about conduits 64, 66, 82. Sampling device 68 may bepreassembled or connected to collection conduit 82. This entirepreassembled structure is then inserted into reaction port 60 and ring76 is secured to threaded portion 78 below rim 74, which definesreaction port 60, to complete the assembly of reaction assembly 62 inreaction port 60.

Finally, harvesting outlet 92 may be inserted in harvesting port 90 toenclose and seal the harvesting port 90. As with the mixing apparatus 32and reaction assembly 62 discussed previously, harvesting outlet 92 isdesirably preassembled, typically with sanitary valve 110 connected tothe harvesting outlet 92 so that the entire harvesting outlet 92 andsanitary valve 110 structure may be inserted into harvesting port 90.Cover 102 associated with harvesting outlet 92 is secured to threadedportion 108 below rim 104, which defines harvesting port 90, to completethe assembly of harvesting outlet 92 in harvesting port 90.

With bioreactor 10 now assembled, reaction medium R may be introducedinto reaction vessel 20 to accomplish the intended biological and/orbiochemical reaction. The vessel 20 may be pre-sterilized by theabove-mentioned techniques while empty, and reaction medium R may beaseptically introduced through a sterilizing grade filter, or reactionmedium R can be sterilized via above-described methods if the reactionmedium R can withstand the sterilization process. Reaction vessel 20 maybe maintained at a predetermined temperature by operation of waterjacket 24. The intended biological and/or biochemical reaction may becarried out in reaction vessel 20 in the presence of one or moremicroorganisms or one or more chemical substances such as, for example,enzymes, under agitation/stirring action of agitator 34. Throughrotation of reaction medium R in reaction vessel 20, the reaction mediumR is treated to effect dispersion and mixing of constituentreactants/substances. In addition, reaction medium R may be furtheraerated through gas introduction conduit 64 which may be necessary forcell growth, as discussed previously. At various intervals duringprocessing, samples of reaction medium R may be taken to monitor theprocessing of the reaction medium R. Such sampling of reaction medium Rwas detailed previously. Additional substances, such as essentialnutrients to support cultivation, may be introduced to reaction medium Rthrough introduction conduit 66 which is part of the reaction assembly62. Once it is determined that reaction medium R is ready for furtherprocessing, the reaction medium R may be transferred to scale-upprocessing in system 200, which includes several scale-up vessels ormain bioreactors 204, 206, 208. The transfer of reaction medium R isaccomplished through harvesting sanitary valve 110, for example, withthe aid of a transfer pump (not shown) in input line 202. Sterileaddition lines and harvest lines should be connected aseptically byusing alcohol or other sterilizing grade chemical to minimizecontamination. The tubing used to transfer the reaction medium R to mainbioreactors 204, 206, 208 should also be pre-sterilized via methodsdescribed previously to minimize introduction of contaminants during thetransfer process.

It may also be preferable to control operation of “seed” bioreactor 10,including agitator 34 and water jacket 24, and the introduction of thereactants and gas to reaction medium R, via a control unit (not shown)such as a computer. In addition, reactant medium R may be removed underthe control of the control unit to transfer the reaction medium R to oneor more of main bioreactors 204, 206, 208. For example, in the case ofcontrol of water jacket 24, the control unit may receive temperatureinput data from temperature sensors in reaction vessel 20 and may adjustoperation of water jacket 24 to control the temperature within reactionvessel 20 within a prescribed temperature range. Similarly, the controlunit may control the fixed delivery of reactant material to reactionvessel 20 by controlling a feed pump connected to introduction conduit66 associated with reaction assembly 62. Further, the control unit couldalso control the supply of gas from a gas bomb connected to gasintroduction conduit 64, for example, by controlling a control valvedisposed between the gas bomb and gas introduction conduit 64. Stillfurther, the control unit could also control the supply of acid/base,gas, nutrients, etc. through the use of an internal measuring devicemonitoring the parameter to be controlled.

While the present invention was described by way of a detaileddescription of several embodiments of a fluid injection apparatus andadaptor pump therefor, those skilled in the art may make modificationsand alterations to this invention without departing from the scope andspirit of the invention. Accordingly, the foregoing description isintended to be illustrative rather than restrictive. The inventiondescribed hereinabove is defined by the appended claims, and all changesto the invention that fall within the meaning and the range ofequivalency of the claims are to be embraced within their scope.

1. A bioreactor apparatus for performing biological and biochemicalreactions comprising: a vessel comprising a mixing port, a reactionport, and a harvesting port; an agitator extending through the mixingport into the vessel; a reaction assembly extending through the reactionport into the vessel, the reaction assembly comprising: a gas conduitadapted to introduce gas into a reaction medium in the vessel; asampling device adapted to remove a portion of the reaction medium fromthe vessel without contamination of the remaining reaction medium; andan introduction conduit permitting the introduction of at least thereaction medium into the vessel; and a harvesting outlet extendingthrough the harvesting port permitting the withdrawal of the reactionmedium to another vessel.
 2. The bioreactor apparatus of claim 1,wherein the bioreactor further comprises a water jacket adapted toregulate the temperature of the reaction medium.
 3. The bioreactorapparatus of claim 1, wherein the gas conduit is adapted to aerate thereaction medium.
 4. The bioreactor apparatus of claim 1, wherein theagitator extends through a cover sealing the mixing port.
 5. Thebioreactor apparatus of claim 1, wherein the agitator is adapted to becoupled to an external drive device.
 6. The bioreactor apparatus ofclaim 5, wherein the external drive device is a motor.
 7. The bioreactorapparatus of claim 1, further comprising a sparger provided in the gasconduit of the reaction assembly to limit the bubble size of the gasintroduced into the vessel.
 8. The bioreactor apparatus of claim 1,wherein the sampling device comprises: a collection conduit extendinginto the vessel; a sampling valve positioned in the collection conduitand disposed outside the vessel; and an extractor associated with thecollection conduit and disposed outside of the vessel and adapted toremove a sample portion of the reaction medium through the collectionconduit.
 9. The bioreactor apparatus of claim 8, further comprising adischarge outlet for releasing the sample portion of the reaction mediumfrom the collection conduit without contamination of the remainingreaction medium.
 10. The bioreactor apparatus of claim 9, wherein theextractor further comprises: a vacuum generating portion adapted todecrease pressurization in the collection conduit; and a filter elementprovided fluidly between the vacuum generating portion and thecollection conduit.
 11. A bioreactor system for performing biologicaland biochemical reactions comprising: a seed bioreactor comprising: avessel comprising a mixing port, a reaction port, and a harvesting port;an agitator extending through the mixing port into the vessel; and areaction assembly extending through the reaction port into the vessel,the reaction assembly comprising: a gas conduit adapted to introduce gasinto a reaction medium in the vessel; a sampling device adapted toremove a portion of the reaction medium from the vessel withoutcontamination of the remaining reaction medium; and an introductionconduit permitting the introduction of at least the reaction medium intothe vessel; a harvesting outlet extending through the harvesting portpermitting the withdrawal of the reaction medium; and a main bioreactoradapted to perform biological and biochemical reactions and adapted tobe placed in fluid communication with the vessel via the harvestingoutlet.
 12. The bioreactor system of claim 11, wherein the seedbioreactor further comprises a water jacket adapted to regulate thetemperature of the reaction medium.
 13. The bioreactor system of claim11, wherein the agitator extends through a cover sealing the mixingport.
 14. The bioreactor system of claim 11, wherein the agitator isadapted to be coupled to an external drive device.
 15. The bioreactorsystem of claim 11, further comprising a sparger provided in the gasconduit of the reaction assembly to limit the bubble size of the gasintroduced into the vessel.
 16. The bioreactor system of claim 11,wherein the sampling device comprises: a collection conduit extendinginto the vessel; a sampling valve positioned in the collection conduitoutside the vessel; an extractor positioned associated with thecollection conduit outside of the vessel and adapted to remove a sampleportion of the reaction medium through the collection conduit; and adischarge outlet for releasing the sample portion of the reaction mediumfrom the collection conduit without contamination of the remainingreaction medium.
 17. The bioreactor system of claim 16, wherein theextractor further comprises: a vacuum generating portion adapted todecrease pressurization in the collection conduit; and a filter elementprovided fluidly between the vacuum generating portion and collectionconduit.
 18. The apparatus of claim 11, wherein the harvesting outletextends through a cover sealing the harvesting port.
 19. The apparatusof claim 11, wherein the main bioreactor comprises an inlet port, andwherein the main bioreactor is placed in fluid communication with thevessel via a sanitary connection between the harvesting outlet and theinlet port.
 20. A method of assembling a bioreactor adapted to performbiological and biochemical reactions comprising: providing a vesselcomprising a mixing port, a reaction port, and a harvesting port;inserting an agitator into the vessel through the mixing port, whereinthe agitator is adapted to stir a reaction medium in the vessel; sealingthe mixing port with a cover, wherein the agitator extends through thecover; sealing the reaction port with a reaction assembly extendingthrough the reaction port and into the vessel; and sealing theharvesting port with a harvesting outlet adapted to permit the sterilewithdrawal and transfer of the reaction medium to another vessel, thereaction assembly comprises: a gas conduit adapted to introduce gas intothe vessel; a sampling device adapted to remove a portion of thereaction medium from the vessel without contamination of the remainingreaction medium; and an introduction conduit permitting the introductionof at least the reaction medium into the vessel.